Method of producing nano- and microcapsules of spider silk protein

ABSTRACT

The present invention is directed to a method of producing nano- and microcapsules from spider silk proteins The invention is further directed to nano- or microcapsules obtainable by this method as well as pharmaceutical, cosmetical and food compositions containing same.

This application is a divisional application of U.S. application Ser.No. 11/989,907, filed Nov. 13, 2009, which is a 371 national stagepatent application of international patent application no.PCT/EP2006/007608 filed Aug. 1, 2006, which claims priority to Europeanpatent application no. 05016712.1, filed Aug. 1, 2005, all of which areincorporated herein by reference in their entireties for all purposes.

The present invention is directed to a method of producing nano- andmicrocapsules from spider silk proteins. The invention is furtherdirected to nano- or microcapsules obtainable by this method as well aspharmaceutical, cosmetical and food compositions containing same.

Small-scale structures are of great interest as transport vesicles andas potential building blocks for future devices. One task is to be ableto encapsulate reactants or particles at small scales and to allowtriggered release of the encapsulated matter after placing them at aspecific location. One solution for such problem is the use of chemicalvesicles, so called nano-capsules. The nano-capsules are designed to setthe reactants free upon an external trigger or stimulus. Severalproblems arise from such quest: the most important one is how to buildsuch nano-capsules in a defined way around e.g. chemically orbiologically active reactants.

Recently, “hybrid” stimuli-responsive nano-capsules have been developedto fulfill such needs. The structures (vesicles but also micelles) areobtained from the self-assembly of e.g. ampihiphile polybutadiene(PB)-b-poly(glutamic acid) (PGA) diblock copolymers, which have apH-sensitive conformation. The pH-sensitivity can be used to unload thevesicles. Those PB-b-PGA copolymers bearing a cross-linkable hydrophobicblock and a hydrophilic peptidic block have been synthesized bycombining anionic and ring-opening polymerization (Chécot et al., 2002).The polydispersity of the copolymers is small enough to obtain welldefined self-assembled aggregates. For example a PB40-b-PGA100 copolymerwhen in water forms closed bilayer vesicles called polymersomes (Won etal., 1999). One property of the vesicles is that they respond to anexternal pH shift by changing in size (FIG. 1). This transition upon pHchanges is reversible and only moderately sensitive to salinity, sinceit is not based on a simple polyelectrolyte swelling effect, but on thepeptidic nature of the PGA block (FIG. 1). These vesicles are not onlyable to encapsulate low molecular weight compounds (like solventmolecules such as fluorophors (Chécot et al., 2003)), but can alsostabilize larger nanoparticles. The disadvantage of such systems is thepartial incompatibility with biological systems, which usually arehighly sensitive to dramatic pH changes, since pH changes can result ina loss in biological activity of the encapsulated sample.

FIG. 1 for illustration shows (a) Dynamic Light Scattering of thepeptosome's hydrodynamic radius R_(H) as a function of NaClconcentration and pH. (b) Schematic representation of the peptosome andits change in size as function of pH due to a coil to α-helix secondarystructure transition in the peptide part.

Another established encapsulation method is the self-assembly ofcolloidal particles at the oil/water interphase. The driving force forthe self-assembly process is the minimization of the total surfaceenergy—thus a wide variety of particles and solvents can be used. Suchstabilized emulsions are well known as Pickering emulsions. Thestabilization or crosslinking of the particles leads to mechanicalstable cages, which can than be transferred to the continuous phase. Theadvantages of the so called colloidosomes are the control of theencapsulate and the ease of tuning the mechanical and chemical stabilityof the outer shell. The self-assembly of the particles results in analmost crystalline structure and thus holes between the particles willoccur. These holes are a size selective filter which allows the controlof the diffusion across the membrane (Dinsmore et al., 2000). The wholeprocess can be performed in a biocompatible way. However, the colloidalparticles themselves are not necessarily biocompatible.

WO 02/47665 describes a method for making self-assembled, selectivelypermeable elastic microscopic structures, referred to as colloidosomes,that have controlled pore-size, porosity and advantageous mechanicalproperties. The method comprises: (a) providing particles formed from abiocompatible material in a first solvent; (b) forming an emulsion byadding a first fluid to said first solvent, said emulsion being definedby droplets of said first fluid surrounded by said first solvent; (c)coating the surface of said droplets with said particles; and (d)stabilizing said particles on said surface of said droplet to formcolloidosomes having a yield strength of at least about 20 Pascals. WO02/47665 uses biocompatible synthetic polymers for producing thesecolloidosomes. Examples are polystyrene, polymethylmethacrylate,polyalkylenes, silica and combinations thereof. The particles from whichthe colloidosomes are to be obtained are stabilized for example bysintering, chemical crosslinking and the like. However, the method ofpreparing those colloidosomes is comparably difficult and the colloidalparticles used may show harmful properties for in vivo applications dueto their artificial and non natural nature. By using colloidal particleslimits also the size range of the shells, since using colloids limitsthe minimal size bag with defined holes.

Therefore, it is a problem underlying the present invention to providenano- and microcapsules, which are highly biocompatible and thussuitable for in vivo applications. It is another problem of thisinvention to obtain nano- and microcapsules, which are capable toaccommodate different types and varying amounts of effective agents ornutritions etc. A further problem underlying the present invention is toprovide nano- and microcapsules which are biodegradable, i.e. which arecapable of a controlled release of said effective agents etc. in vivo,for example in topical or systemic applications.

These problems are solved by the subject-matter of the independentclaims. Preferred embodiments are set forth in the dependent claims.

In the present invention, it surprisingly turned out that spider silkproteins may serve as a base for forming micro- and nanocapsules whichcan be used for various in vivo applications. It in particular turnedout that this can be done by an improved method of producing saidcapsules, which does circumvent the need of using steps to link orstabilize the particles from which the capsules are formed by additionof chemicals like crosslinkers or which requires sintering or the likewhich could have deleterious effects on the agents to be packaged intosaid micro- and nanocapsules.

Since most currently used encapsulation techniques (see for example WO02/47665) rely on non-biological particles or macromolecules, theinventors developed a new stable encapsulation process based onself-assembling spider silk proteins. Unlike other encapsulationtechnologies, in the present method the hydrophobic/hydrophilic natureof the emulsion surface is not only used to assemble colloid particles,but it is also used as the driving force for the colloid immobilizationthrough coalescence and polymer network formation (stabilization). Thisprocess represents not only a method to produce polymeric nano- andmicrocapsules formed from a new class of biocompatible colloids, it alsorepresents a novel approach to polymer network formation using proteins.The big advantage of nano- and microcapsules formed from this method isthe biocompatibility and the functionality of the microcapsules impartedby the proteins. This enables the control of the release mechanisms byseveral means: pH changes, temperature changes, or activity ofproteases.

For example, the nano- or microcapsules might be destroyed and theiringredients might be released in vivo chemically, physically (forexample by shear forces) or biologically (by proteolytic digestion).

The self-assembly of the spider silk proteins at the interface wasachieved by introducing the protein into the water phase of a water/oilemulsion (see FIG. 2). The minimization of surface energy was drivingthe proteins to the interface and induced an aggregation of the monomersto a dense polymer network (FIG. 3).

The spider bags/balloons formed from this process are for example filledwith the contents of the water phase and can exist in organic solvents,alcohols as well as in water (FIG. 3). Therefore, they are showing anunexpected stability in strongly differing environments. In principalthe self-assembly of proteins at an inverse emulsion surface is alsopossible-thus encapsulating the content of the oil phase (see alsobelow).

Strikingly, the bags/balloons can be filled with proteins, chemicalreactants, nano- and micrometer scaled particles, etc., which isexemplarily shown by filling the particles with fluorescently(FITC)-labeled Dextran particles (FIG. 4).

The impermeability of the membrane and the mechanical stability of thebags against osmotic stresses are both relatively high, considering thethickness of the membrane. Electron microscopy images reveal that thethickness is between 10 and 70 nm (FIG. 5).

In the present approach synthetic spider silk proteins have beenemployed, in particular the synthetic sequence of C₁₆ (Huemmerich etal., 2004) to create a biological encapsulation of active agents.

Spider silks in general are protein polymers that display extraordinaryphysical properties, but there is only limited information on thecomposition of the various silks produced by different spiders (seeScheibel, 2004). Among the different types of spider silks, draglinesfrom the golden orb weaver Nephila clavipes and the garden cross spiderAraneus diadematus are most intensely studied. Dragline silks aregenerally composed of two major proteins and it remains unclear whetheradditional proteins play a significant role in silk assembly and thefinal silk structure. The two major protein components of draglines fromAraneus diadematus are ADF-3 and ADF-4 (Araneus Diadematus Fibroin).

Genes coding for spider silk-like proteins were generated using acloning strategy, which was based on a combination of synthetic DNAmodules and PCR-amplified authentic gene sequences (Huemmerich et al.,2004). The dragline silk proteins ADF-3 and ADF-4 from the garden spiderAraneus diadematus were chosen as templates for the syntheticconstructs. A seamless cloning strategy allowed controlled combinationof different synthetic DNA modules as well as authentic gene fragments.A cloning vector was designed comprising a cloning cassette with aspacer acting as placeholder for synthetic genes (Huemmerich et al.,2004).

To mimic the repetitive sequence of ADF-4 a single conserved repeat unithas been designed to gain one consensus module termed C, which wasmultimerized to obtain the repetitive protein C₁₆, which was employed inthe given approach as an example.

There are many possible applications for the presented spider silkbags/balloons, ranging from functional food to pharmaceutical tocosmetical applications. For example the encapsulation in foodtechnology could protect certain ingredients such as vitamins from anoxidizing environment. In another food technology application,ingredients such as fish oil could be hidden from taste. Inpharmaceutical applications the diffusion barrier of the protein shellallows for slow (controlled) release processes for the encapsulatedmaterial. The further design of the protein shells could result in adefined release container, which liberates the content only afteractivation using certain proteases or other triggers. In cosmetics thetransport of water active ingredients into the skin could be facilitatedby the presented bags/balloons, after slow degradation of the proteinshell, e.g. by proteases of the skin. Further, mechanical shearing canbe used to liberate the content upon exposure to the skin.

The present invention in particular is directed to the following aspectsand embodiments:

According to a first aspect, the present invention is directed to amethod of producing nano- and microcapsules comprising the steps of:

-   a) providing spider silk proteins;-   b) forming a solution or suspension of said proteins in a suitable    solvent;-   c) generating an emulsion of at least two phases, said emulsion    containing the solution or suspension formed in b) as a first phase    and at least one further phase, which is substantially immiscible    with said first phase;-   d) forming a polymer network of the spider silk proteins at the    interface of the at least two phases;-   e) separating the protein polymer network generated in (d) from the    emulsion.

As explained above, it unexpectedly turned out that forming the polymernetwork in step d) does not require the addition of any furtheringredients (for example crosslinkers) and there is no need foradditional steps as sintering, crosslinking etc.

It is noted that the term “spider silk protein” as used herein does notonly comprise all natural sequences but also all artificial or syntheticsequences which were derived therefrom.

Accordingly, the spider silk sequences may be derived from sequenceswhich are termed “authentic” herein. This term means that the underlyingnucleic acid sequences are isolated from their natural environmentwithout performing substantial amendments in the sequence itself. Theonly modification, which is accepted to occur, is where the authenticnucleic acid sequence is modified in order to adapt said sequence to theexpression in a host without changing the encoded amino acid sequence.Preferred sequences are NR3 (SEQ ID NO: 10; derived from ADF-3) and NR4(SEQ ID NO: 11; derived from ADF-4). In both sequences, for moreefficient translation, the codon AGA (Arg), which is rarely translatedin E. coli, was mutated to CGT (Arg) using PCR mutagenesis.

The authentic non-repetitive sequences are preferably derived from theamino terminal non-repetitive region (flagelliform proteins) and/or thecarboxy terminal non-repetitive region (flagelliform and draglineproteins) of a naturally occurring spider silk protein. Preferredexamples of those proteins will be indicated below.

According to a further embodiment, the authentic non-repetitivesequences are derived from the amino terminal non-repetitive region(flagelliform proteins) and/or the carboxy terminal non-repetitiveregion (flagelliform and dragline proteins) of a naturally occurringspider silk protein.

Preferred authentic sequences of flagelliform proteins are the aminoacid sequence and nucleic acid sequence of FlagN-NR (SEQ ID NOs: 31 and32) and FlagC-NR (SEQ ID NOs: 33 and 34).

The recombinant spider silk proteins of the invention generally may bederived from spider dragline proteins from the spider's major ampullategland and/or from proteins derived from the flagelliform gland.

According to an embodiment, the recombinant (synthetic/artificial)spider silk proteins which can be used in the present inventiongenerally are derived from spider dragline proteins from the spider'smajor ampullate gland and/or from proteins derived from the flagelliformgland.

It is generally preferred to select the dragline and/or flagelliformsequences from dragline or flagelliform proteins of orb-web spiders(Araneidae and Araneoids).

More preferably the dragline proteins and/or flagelliform proteins arederived from one or more of the following spiders: Arachnura higginsi,Araneus circulissparsus, Araneus diadematus, Argiope picta, BandedGarden Spider (Argiope trifasciata), Batik Golden Web Spider (Nephilaantipodiana), Beccari's Tent Spider (Cyrtophora beccarii), Bird-droppingSpider (Celaenia excavata), Black-and-White Spiny Spider (Gasteracanthakuhlii), Black-and-yellow Garden Spider (Argiope aurantia), Bolas Spider(Ordgarius furcatus), Bolas Spiders-Magnificent Spider (Ordgariusmagnificus), Brown Sailor Spider (Neoscona nautica), Brown-Legged Spider(Neoscona rufofemorata), Capped Black-Headed Spider (Zygiellacalyptrata), Common Garden Spider (Parawixia dehaani), Common Orb Weaver(Neoscona oxancensis), Crab-like Spiny Orb Weaver (Gasteracanthacancriformis (elipsoides)), Curved Spiny Spider (Gasteracantha arcuata),Cyrtophora moluccensis, Cyrtophora parnasia, Dolophones conifera,Dolophones turrigera, Doria's Spiny Spider (Gasteracantha doriae),Double-Spotted Spiny Spider (Gasteracantha mammosa), Double-Tailed TentSpider (Cyrtophora exanthematica), Aculeperia ceropegia, Eriophorapustulosa, Flat Anepsion (Anepsion depressium), Four-spined Jewel Spider(Gasteracantha quadrispinosa), Garden Orb Web Spider (Eriophoratransmarina), Giant Lichen Orbweaver (Araneus bicentenarius), Golden WebSpider (Nephila maculata), Hasselt's Spiny Spider (Gasteracanthahasseltii), Tegenaria atrica, Heurodes turrita, Island Cyclosa Spider(Cyclosa insulania), Jewel or Spiny Spider (Astracantha minax), KidneyGarden Spider (Araneus mitificus), Laglaise's Garden Spider (Enrovixialaglaisei), Long-Bellied Cyclosa Spider (Cyclosa bifida), Malabar Spider(Nephilengys malabarensis), Multi-Coloured St Andrew's Cross Spider(Argiope versicolor), Ornamental Tree-Trunk Spider (Herenniaornatissima), Oval St. Andrew's Cross Spider (Argiope aemula), Red TentSpider (Cyrtophora unicolor), Russian Tent Spider (Cyrtophora hirta),Saint Andrew's Cross Spider (Argiope keyserlingi), Scarlet Acusilas(Acusilas coccineus), Silver Argiope (Argiope argentata), SpinybackedOrbweaver (Gasteracantha cancriformis), Spotted Orbweaver (Neosconadomiciliorum), St. Andrews Cross (Argiope aetheria), St. Andrew's CrossSpider (Argiope Keyserlingi), Tree-Stump Spider (Poltys illepidus),Triangular Spider (Arkys clavatus), Triangular Spider (Arkyslancearius), Two-spined Spider (Poecilopachys australasia), Nephilaspecies, e.g. Nephila clavipes, Nephila senegalensis, Nephilamadagascariensis and many more (for further spider species, see alsobelow). Most preferred, the dragline proteins are derived from Araneusdiadematus and the flagelliform proteins are derived from Nephilaclavipes.

In the context of this invention, it should be clear that a recombinantspider silk protein may not only comprise protein sequences from onespecies, but may also contain sequences derived from different spiderspecies. As an example, the one or more synthetic repetitive spider silkprotein sequences might be derived from one species, the one or moreauthentic non-repetitive spider silk protein sequences from another. Asa further example, it is also possible to design a recombinant spidersilk protein, which contains more than one type of a repetitivesequence, wherein the different types are derived from differentspecies.

According to one preferred embodiment, the dragline protein is wild typeADF-3, ADF-4, MaSp I, MaSp II and the flagelliform protein is FLAG. Theterm ADF-3/-4 is used in the context of MaSp proteins produced byAraneus diadematus (Araneus diadematus fibroin-3/-4). Both proteins,ADF-3 and -4 belong to the class of MaSp II proteins (major ampullatespidroin II).

In a further embodiment, the nucleic acid sequence provided is ADF-3(SEQ ID NO:1) and/or ADF-4 (SEQ ID NO: 2), or a variant thereof.

It is noted that two different kinds of ADF-3 and ADF-4 coding sequencesand proteins are contemplated in this invention: first, the alreadypublished sequence of ADF-3 and ADF-4 (herein: “wild type” sequence)and, second, a variant thereof, encoded by SEQ ID NO: 1 (ADF-3) and 2(ADF-4). The wild type sequences were already published and areavailable under the accession numbers U47855 and U47856 (SEQ ID NO: 8and 9).

Further spider silk proteins, which can be used in this invention (i.e.alone or in combination with further proteins) and their databaseaccession numbers are:

-   spidroin 2 [Araneus bicentenarius]gi|2911272-   major ampullate gland dragline silk protein-1 [Araneus    ventricosus]gi|27228957-   major ampullate gland dragline silk protein-2 [Araneus    ventricosus]gi|27228959 ampullate spidroin 1-   [Nephila madagascariensis]gi|13562006-   major ampullate spidroin 1 [Nephila senegalensis]gi|13562010-   major ampullate spidroin 1 [Latrodectus geometricus]gi|13561998-   major ampullate spidroin 1 [Argiope trifasciata]gi|13561984-   major ampullate spidroin 1 [Argiope aurantia]gi|13561976-   dragline silk protein spidroin 2 [Nephila clavata]gi|16974791-   major ampullate spidroin 2 [Nephila senegalensis]gi|13562012-   major ampullate spidroin 2 [Nephila madagascariensis]gi|13562008-   major ampullate spidroin 2 [Latrodectus geometricus]gi|13562002

According to another preferred embodiment, the flagelliform protein isSEQ ID NO: 6 (Flag-N) and/or SEQ ID NO: 7 (Flag-C) or a variant thereof.

However, also already known and published flagelliform sequences may beused herein, in particular the following:

-   Flagelliform silk protein partial cds [Nephila clavipes]gi|2833646-   Flagelliform silk protein partial cds [Nephila clavipes]gi|2833648

In one preferred embodiment, the recombinant spider silk proteincomprises one or more synthetic repetitive sequences containing one ormore polyalanine containing consensus sequences. Those polyalaninesequences may contain from 6 to 9 alanine residues. See, for example SEQID NO: 1, containing several polyalanine motifs of 6 alanine residues.

Preferably, the polyalanine containing consensus sequence is derivedfrom ADF-3 and has the amino acid sequence of SEQ ID NO: 3 (module A) ora variant thereof. Module A contains a polyalanine having 6 alanineresidues. A further preferred polyalanine containing consensus sequence,derived from ADF-4, is module C (SEQ ID NO: 5), containing 8 alanineresidues.

According to a further preferred embodiment, in the recombinant spidersilk protein of the invention, the synthetic repetitive sequence isderived from ADF-3 and comprises one or more repeats of the amino acidsequence of SEQ ID NO: 4 (module Q) or a variant thereof.

In more general words, a synthetic repetitive sequence may also containthe general motifs: GGX or GPGXX, i.e. glycine rich regions. Asmentioned above, these regions will provide flexibility to the proteinand thus, to the thread formed from the recombinant spider silk proteincontaining said motifs.

It is noted that the specific modules for the synthetic repetitivesequence for use in the present invention can also be combined with eachother, i.e. modules (repeat units) combining A and Q, Q and C etc. arealso encompassed by the present invention. Although the number of themodules to be introduced in the spider silk protein is not restricted,it is preferred to employ a number of modules of the syntheticrepetitive sequence for each recombinant protein which number ispreferably ranging from 5-50 modules, more preferably 10-40 and mostpreferably between 15-35 modules.

The synthetic repetitive sequence preferably comprises one or more of(AQ) and/or (QAQ) as repeat units. Even more preferred, the syntheticrepetitive sequence is (AQ)₁₂, (AQ)₂₄, (QAQ)₈ or (QAQ)₁₆.

Whenever the synthetic repetitive sequence is derived from ADF-4, it maypreferably comprise one or more repeats of the amino acid sequence ofSEQ ID NO: 5 (module C) or a variant thereof, as mentioned above,wherein the overall synthetic repetitive sequence is C₁₆ or C₃₂.

Preferred embodiments for the complete recombinant spider silk proteinsof the invention are (QAQ)₈NR3, (QAQ)₁₆NR3, (AQ)₁₂NR3, (AQ)₂₄NR3, C₁₆NR4and C₃₂NR4 i.e. proteins which comprise or consist of said sequences.

It is noted that the above configuration of the synthetic repetitivesequence (using the A, Q and C system) also applies to all other repeatunits disclosed above, for example all polyalanine containing sequencescan be taken for A and/or C and all glycine rich sequences may be usedas Q.

New modules for synthetic repetitive sequences derived from flagelliformsequences are modules K (SEQ ID NO: 35 and 36), sp (SEQ ID NO: 37 and38), X (SEQ ID NO: 39 and 40), and Y (SEQ ID NO: 41 and 42):

The synthetic repetitive sequence also preferably comprises or consistsof Y₈, Y₁₆, X₈, X₁₆, K₈, K₁₆. Furthermore, it is also possible, tocombine those sequences derived from ADF-3 and ADF-4 and Flag in onerecombinant sequence.

In the present invention it is however strongly preferred to employspider silk proteins in step a) which are selected from or containingsequences of the group of ADF-4 sequences and derivatives thereofincluding C₁₆, C₁₆NR4, C₃₂ and/or C₃₂NR4.

In the present invention the spider silk proteins can be furtherengineered to contain single amino acid substitutions or direct chemicalmodifications before capsule production, or the latter also aftercapsule production. This can be used to introduce e.g. specific bindingaffinities to the bags or to introduce protease specific amino acidsequences. This may result in a controlled release of the encapsulate byproteolytic digestion of the silk membrane.

By introducing e.g. single cysteines crosslinking of the bag or thecovalent coupling of different functional groups can be achieved. Forexample, replacing nucleic acids encoding one or more amino acids in aspider silk protein by a lysine or cysteine encoding nucleic acidsequence, and/or adding a nucleic acid sequence containing nucleic acidsencoding lysine and/or cysteine to said sequence, may achieve this.

Further, agents may be coupled to the spider silk proteins before andafter formation of the nano- or microcapsules in order to direct thecapsules to specific cells or tissues. This can be achieved, forexample, by introducing or covalent coupling of specific RGD sequences.Thus, RGD peptides may be cross-linked to the spider silk proteinsbefore and after formation of the nano- or microcapsules. Examples forcyclic RGD molecules are indicated in FIG. 10.

Furthermore, cell or tissue specific antibodies and cell or tissuespecific receptors might be coupled to the spider silk proteins todirect the capsules to a specific target.

According to a further embodiment, the solvent in b) and/or the solventsof the at least one further phase is selected from the group consistingof hydrophilic solvents, preferably water, alcohols like ethanol,glycerol, or lipophilic solvents, preferably natural oils, such as oilsof plant or animal origin, synthetic oils, such as miglyol, silicon oil,organic solvents, such as aromatic hydrocarbons, for example toluene,benzene etc.

It is noted that one single phase may contain also more than one solvent(i.e. a mixture) as long as the solvents are substantially identical.“Substantially identical” means that the solvents are having similarsolubility properties thus forming only one common phase. Thus,“substantially identical” solvents include solvents in which one can notobserve separate phases if the solvents are blended. As an example, twoor more lipophilic solvents may be combined into one phase, for examplea plant oil (for example olive oil and castor oil) and miglyol and/orhexadecane. Or, as an alternative, a hydrophilic phase may comprise twoor more hydrophilic components, for example water, glycerol and thelike.

As mentioned above, the only requirement is that the emulsion system forproducing the nano- and microcapsules of the invention has at least twophases, wherein the phases are substantially immiscible.

All known emulsion types may be employed in step c) of the presentmethod, for example W/O, O/W, O/W/O or W/O/W type emulsions. Theseemulsion types are well known in the art and for further information itis for example referred to “Remington's Pharmaceutical Sciences”, MackPublishing Co., Easton, Pa., latest edition or further availableinformation.

A preferred method for forming the emulsions of the present invention isto produce a mini-emulsion. Mini-emulsions are dispersions of criticallystabilized oil droplets with a size between 50 and 500 nm prepared byshearing a system containing oil, water, a surfactant and a hydrophobicagent. Polymerizations in such mini-emulsions, when carefully prepared,result in particles which have about the same size as the initialdroplets. This means that the appropriate formulation of a mini-emulsionsuppresses coalescence of droplets or Ostwald ripening. The preparationof the mini-emulsion is done by high shear devices such as ultrasoundand high-pressure homogenizers. It is referred to the variouspublications of K. Landfester and coworkers.

In case of an emulsion of the W/O type, the W (hydrophilic) phase isforming the emulsion droplets and in this case, the spider silk proteinsare contained in the W phase. The O phase is the lipophilic phase andforms the continuous phase.

In case of an emulsion of the O/W type, the O (lipophilic) phase isforming the emulsion droplets and in this case, the spider silk proteinsare contained in the O phase. The W phase is the hydrophilic phase andforms the continuous phase.

The surfactants used in the above emulsions may be selected from thosecompounds, the skilled person will use based on the available knowledgein the field of pharmaceutical and related sciences. An exemplaryselection of surfactants for use in obtaining the present emulsions arefatty acid esters of glycerols, sorbitol and other multifunctionalalcohols, preferably, glycerol monostearate, sorbitan monolaurate, orsorbitan monoleate; poloxamines; polyoxyethylene ethers andpolyoxyethylene esters; ethoxylated triglycerides; ethoxylated phenolsand ethoxylated diphenols; metal salts of fatty acids, metal salts offatty alcohol sulfates, sodium lauryl sulfate; and metal salts ofsulfosuccinates: polysorbates, more preferably polysorbate 20, 40, 60and 80: poloxamers, polyoxyethylene glycols; and mixtures of saidsubstances.

However, it is explicitly noted that it is not an essential feature ofthis invention to use a surfactant. The skilled artisan is aware ofemulsion systems, which do not require surfactants.

In a preferred embodiment of the present invention, the solvent used in1b) further contains one or more pharmaceutical agents, cosmeticalagents, foodstuffs or food additives. In other words, the additionalingredients usually will be present in the phase, which is alsocontaining the spider silk proteins. In this case, the one or moreingredients/agents will be encapsulated into the polymer network whichis formed at the phase-interface.

As an alternative, it is also possible to add the above mentioned agentsto the continuous phase, which does not contain the spider silkproteins. In this case, the nano- and microcapsules of the inventionwill be coated by said agents.

As a further alternative, the agents may be introduced into the nano-and microcapsules of the invention after they have been obtained by thepresent method.

This can be done by swelling the membrane with certain solvents andletting the encapsulate (effective agent) diffuse inside. Swelling couldalso be done by temperature, pressure or not only solvents but alsoother chemical means (such as chemical agents, pH, and others).

It is also possible to incorporate the encapsulate into the membrane.This approach may give amended or improved release properties thanencapsulating the encapsulate into it.

The type of agent which is additionally incorporated into the nano- andmicrocapsules of the invention is not restricted in any way.

For example, the pharmaceutical agent may be selected from the groupconsisting of analgesics; hypnotics and sedatives; drugs for thetreatment of psychiatric disorders such as depression and schizophrenia;anti-epileptics and anticonvulsants; drugs for the treatment ofParkinson's and Huntington's disease, aging and Alzheimer's disease;drugs aimed at the treatment of CNS trauma or stroke; drugs for thetreatment of addiction and drug abuse; chemotherapeutic agents forparasitic infections and diseases caused by microbes; immunosuppressiveagents and anti-cancer drugs; hormones and hormone antagonists;antagonists for non-metallic toxic agents; cytostatic agents for thetreatment of cancer; diagnostic substances for use in medicine;immunoactive and immunoreactive agents; antibiotics; antispasmodics;antihistamines; antinauseants; relaxants; stimulants; cerebral dilators;psychotropics; vascular dilators and constrictors; anti-hypertensives;drugs for migraine treatment; hypnotics, hyperglycemic and hypoglycemicagents; anti-asthmatics; antiviral agents; and mixtures thereof.

Foodstuffs and food additives may be selected from the group consistingof vitamines (ascorbic acid, tocopherol acetate and the like), minerals(calcium, magnesium, potassium, sodium, for example), trace elements(selenium), extracts of natural origin, natural oils (fish oil) etc.

Cosmetical agents may be selected for example from tocopherol acetate,oils of natural or synthetic origin, panthenol, plant extracts, UVabsorbing agents, desinfectants, anti-irritant agents, repellants.

It is noted that the agents might be present in the solvent indissolved, suspended or solid form. In the latter case, a solid core isprovided which is coated by the spider silk proteins of the presentinvention.

In a preferred embodiment, the separation of the polymer network in stepe) is done by means of centrifugation or by destroying the emulsionformed in step c) and forming a one-phase solution. However, also othermethods may be used in order to separate the nano- and microcapsules ofthe present invention from the emulsion system.

The temperature used in steps b)-e) is 5-40° C., preferably 10-30 andmore preferably room temperature. The pH used in steps b)-e) is 3-9,preferably 5-8, more preferably 7.

The size of the emulsion droplets and the nano- and microparticlesderived therefrom is preferably from 10 nm to 40 μm, preferably between500 nm and 10 μm, most preferably about 5 μm. The wall thickness of theobtained nano- and microcapsules preferably is between 5 and 100 nm,more preferably between 10 and 70 nm (see for example FIG. 5).

In a second aspect, the present invention provides nano- andmicrocapsules obtainable by the method as disclosed above.

A third aspect of the present invention is directed to a pharmaceuticalcomposition containing nano- and microcapsules as defined above and oneor more pharmaceutically acceptable carriers. Thus, the activecomponents of the present invention are preferably used in such apharmaceutical composition in doses mixed with an acceptable carrier orcarrier material, that the disease can be treated or at leastalleviated. Such a composition can (in addition to the active componentand the carrier) include filling material, salts, buffer, stabilizers,solubilizers and other materials, which are known state of the art.

The term “pharmaceutically acceptable” is defined as non-toxic material,which does not interfere with effectiveness of the biological activityof the active component. The choice of the carrier is dependent on theapplication.

The pharmaceutical composition can contain additional components whichenhance the activity of the active component or which supplement thetreatment. Such additional components and/or factors can be part of thepharmaceutical composition to achieve a synergistic effects or tominimize adverse or unwanted effects.

Techniques for the formulation or preparation and application/medicationof compounds of the present invention are published in “Remington'sPharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latestedition (see also above). An appropriate application can include forexample oral, dermal or transmucosal application and parenteralapplication, including intramuscular, subcutaneous, intramedularinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal or intranasal injections.

In a fourth aspect, the present invention provides a cosmetical or foodproduct containing nano- and microcapsules as disclosed hereinabove.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The invention is now further illustrated by the accompanying figures, inwhich:

FIG. 1 shows (a) Dynamic Light Scattering of the peptosome'shydrodynamic radius R_(H), as a function of NaCl concentration and pH.(b) Schematic representation of the peptosome and its change in size asfunction of pH due to a coil to α-helix secondary structure transitionin the peptide part.

FIG. 2 is an schematic illustration of the spider bag/balloon formationprocess. (A) An aqueous protein suspension is emulsified in toluene. (B)Protein adsorbs at the water-toluene interface and denatures forming apolymer network (Inset). (C) Once adsorbed, the protein network can betransferred into water by centrifugation. The final bag/balloonstructures have water on the inside and water on the outside. (D)Alternatively, once adsorbed, the protein network can be transferredinto a one-phase solution through the addition of ethanol.

FIG. 3 shows an image of spider bags/balloons in (A) toluene/ethanol(50:50) and (B) after transfer into water.

FIG. 4 is an image of spider bags/balloons filled with FITC-labeledDextran (MW 500 kDa) after transfer into the continuous water phase: (A)bright field image. (B) fluorescent image.

FIG. 5 shows dried Spider bags/balloons imaged by SEM. The membranethickness has been determined to be smaller than 70 nm n.

FIG. 6 IR spectra in D₂O before ( - - - . - - - .) and after ( - - - )microcapsule formation. The shift in the spectra indicates a change inprotein structure during microcapsule formation. Specifically, thisshift indicates the formation of β-sheets. Deconvolution and Gaussianfit of microcapsule IR spectra reveals four peaks. Deconvoluted peaksare at 1621 cm⁻¹, 1642 cm⁻¹, 1661 cm⁻¹ and 1683 cm⁻¹ and are assignedrespectively to β-sheets, random coil structures and two peaks forβ-turns.

FIG. 7 Microcapsule permeability measurements. (A) Sample confocal imageof a microcapsule in water with 0.3% 4 kDa FITC dextran added outsidethe microcapsule. A fraction of the dextran permeates the membrane. (B)Molecular weight distribution of dextran as measured by GPCfluorescence. (C) Measured molecular weight cutoff histogram for 51microcapsules. Average molecular weight cutoff is 2.2 kDa.

FIG. 8 Proteinase K digestion of C₁₆ microcapsules. Top row phasecontrast image. Bottom row fluorescent image. As indicated by loss offluorescence, dextran is released shortly after Proteinase K addition.Complete digestion of microcapsules occurs after 13±1 minutes.

FIG. 9 Applied force versus microcapsule deformation as measured by AFM.Inset graph is the linear force regime during small deformations (ε<1%).

FIG. 10A and FIG. 10B illustrate the final steps in the synthesis ofcyclic RGD molecules for use in the present invention.

EXAMPLES

Protein Preparation

The protein solution, from which the spider balloons were formed, wasprepared by first dissolving recombinant spider dragline silk protein(C₁₆, see Huemmerich et al., 2004) at a concentration of 10 mg/ml in 6Mguanidine thiocyanate. The protein solution was cooled to 4° C. and theconcentration of guanidine thiocyanate was reduced below 1 mM bydialyzing the protein solution against a 10 mM Tris buffer, pH 8.0overnight using dialysis tubing from Carl Roth GmbH with a molecularweight cutoff of 14 kDa. Any undispersed protein was removed bycentrifuging the dialyzed solution for 30 minutes at a force of100.000×g while maintaining the solution temperature at 4° C. The finalprotein concentration was determined using UV adsorption, employing theproteins extinction coefficient of 0.859 at a wavelength of 276 nm.

Microcapsule Formation

Microcapsules of spider silk were formed by emulsifying 5 μl of dialyzedprotein suspension in 300 μl toluene for 90 seconds (FIG. 2A). Duringemulsification, silk protein adsorbs and changes its structuralconformation at the surface of the emulsion droplets resulting in apolymer network that encapsulates the emulsion droplet (FIG. 2B). Spidersilk microcapsules were formed using protein suspensions withconcentrations ranging from 1 to 6 mg/ml and with emulsification timesas short as 20 seconds. The size of the microcapsules formed depends onthe size of the emulsion droplets.

Once formed, the protein shells surrounding the emulsion droplets weretransferred from the two-phase emulsion into a one-phase solution. Twodifferent methods are effective in transferring the protein shells. Inthe first method, 300 μl of water was added to the toluene to form anaqueous sublayer. The protein shells surrounding the water droplets werecentrifuged from the toluene layer into the aqueous sublayer at a forceof 100×g for 4 minutes (FIG. 2C). In the second method, a one-phasesolution was formed by adding 300 μl of ethanol to the two-phaseemulsion, in order to solubilize the toluene and water (FIG. 2D). Afterusing either method to transfer the microcapsules to a one-phasesolution, the resulting structures were investigated with an opticalmicroscope (FIG. 3).

Unlike soluble C₁₆, whose structure is primarily random coil, theassembled protein has a β-sheet-rich conformation. The change in C₁₆conformation upon assembly was observed using IR microscopy. Initially,C₁₆ solubilized in D₂O adsorbs at 1645 cm⁻¹, which is characteristic ofproteins in a random coil structure (FIG. 6). After microcapsuleformation two shoulders in the adsorption spectra emerge indicating achange in the secondary structure of C₁₆. Deconvolution of the spectrareveals the contribution of four Gaussian peaks at 1621 cm⁻¹, 1642 cm⁻¹,1661 cm⁻¹ and 1683 cm¹.

The integrity of the centrifuged microcapsule-like protein shells wasverified by adding 0.5 wt %, FITC labeled, 500 kDa Dextran(Sigma-Aldrich) to the protein solution prior to emulsification. Afteremulsification and centrifugation, the formed microcapsule-likestructures continued to fluoresce indicating that the protein shell ofthese structures did not tear during centrifugation (FIG. 4). Themicrocapsule membrane can trap large molecules such as high molecularweight dextran but is permeable to small molecules such as fluorescein.If low molecular weight FITC labeled dextran is added to the outside ofthe centrifuged microcapsules a fraction of the dextran permeates themembranes and enters the capsules (FIG. 7A). The fractional admittanceof the low molecular weight dextran occurs because the dextran has anon-finite polydispersity, comprising of both low and high dextranmolecules (FIG. 7B). As a result the membrane admits the dextran below acertain molecular weight cutoff and excludes the dextran larger thanthis cutoff. By measuring the amount of fluorescence intensity insidethe microcapsules and by using the fluorescent molecular weightdistribution of the dextran (FIG. 7B) as measured by gel permeationchromatography, the molecular weight cutoff of the membrane wasdetermined. The permeability of 52 different microcapsules in 13different samples were measured. The molecular weight cutoff of thesemicrocapsules ranged from 0.3 kDa to 6.0 k-Da with an average molecularweight cutoff of 2.2 kDa (r_(g)˜18 Å) (FIG. 7C).

Enzymatic triggered release of contents, such as FITC-labeled dextran,was demonstrated using the enzyme Proteinase K (FIG. 8). As indicated bythe loss of fluorescence, shortly after the addition of Proteinase K theintegrity of the microcapsule membrane is destroyed and the dextran isreleased. After the release of the dextran, the enzyme continues todigest the microcapsule until complete digestion occurs at 13±1 minutes.

The enzymatic digestion of the microcapsules can be prevented bychemically cross-linking C₁₆ through photo-initiated oxidation withammonium peroxodisufate (APS) and Tris (2,2′-bipyridyl)dichlororuthenium(II) (Rubpy). To chemically crosslink the C₁₆, 10 mMAPS and 100 mM Rubpy are added to the centrifuged solution, and thereaction is photo-initiated by exposing the mixture to light from atungsten lamp for 5 minutes. This cross-linking renders the C₁₆microcapsules stable against treatment with Proteinase K. Aftercross-linking, the addition of 100 μM Proteinase K to the crosslinkedmicrocapsules has no effect on capsule integrity even after incubationfor one hour at 37° C. This behavior is markedly different from thenon-crosslinked microcapsules which release the encapsulated dextranalmost immediately under the same conditions.

The formed microcapsules are observed to be highly elastic. Theelasticity of the microcapsules was measured by compression with an AFM.For the compression measurements a 35 micron glass sphere attached to anAFM cantilever with a spring constant of 10 pN/nm and force versusdeformation curves were obtained for microcapsules with sizes rangingfrom 1 to 4 microns (FIG. 9). At small deformations the relationshipbetween the applied force, f, and the resultant deformation, ε, isdescribed byf∝Eh ²ε/√{square root over (12(1−σ²))}where h is the membrane thickness, E is the Young's modulus, σ is thePoisson ratio, and the pre-factor is a constant of an order of one.Using the maximum capsule wall thickness calculated from the initialconcentration of silk monomer used and by assuming a Poisson ratio of0.5, the microcapsules were determined to have a Young's modulus betweenE=0.7-3.6 GPa. The capsules also demonstrate excellent chemicalstability. The addition of protein denaturants such as 2% sodiumdodecylsulfate (SDS) and 8M urea has no effect on capsule integrity. Themicrocapsules were observed to be stable under these conditions forweeks.

REFERENCES

-   Chécot F, Lecommandoux S, Gnanou Y, Klok H A (2002) Angew. Chem.    Int. Ed. 41, 1339-   Chécot F, Lecommandoux S, Klok H A, Gnanou Y (2003) Euro. Phys. J. E    10, 25-   Dinsmore A D, Hsu M F, Nikolaides M G, Marquez M, Bausch A R, Weitz    D A. (2002) Colloidosomes: Selectively permeable capsules composed    of colloidal particles. Science 298(5595):1006-1009-   Y. Y. Won, H. Davis, F. Bates, Science 283, 960 (1999)-   Huemmerich D, Helsen C W, Quedzueweit S, Oschmann J, Rudolph R,    Scheibel T (2004) Primary structure elements of spider dragline    silks and their contribution to protein solubility. Biochemistry 43:    13604-12-   Scheibel T (2004) Spider silks: recombinant synthesis, assembly,    spinning, and engineering of synthetic proteins, Microbial Cell    Factories 3, 14

What is claimed is:
 1. A nanocapsule of spider silk proteins, whereinthe spider silk proteins form a polymer network that encapsulates anemulsion droplet.
 2. The nanocapsule of spider silk proteins of claim 1,wherein said nanocapsule has a wall.
 3. The nanocapsule of spider silkproteins of claim 2, wherein the wall thickness of said nanocapsule isbetween 5 and 100 nm.
 4. The nanocapsule of spider silk proteins ofclaim 1, wherein the spider silk proteins comprise 5 to 50 repeat units,wherein the repeat unit is selected from the group consisting of theamino acid sequence according to SEQ ID NO: 3, the amino acid sequenceaccording to SEQ ID NO: 4, and the amino acid sequence according to SEQID NO:
 5. 5. The nanocapsule of spider silk proteins of claim 1, whereinthe spider silk proteins are selected from the group consisting of C₁₆,C₃₂, (AQ)₁₂, (AQ)₂₄, (QAQ)₈ and (QAQ)₁₆, wherein C represents the aminoacid sequence according to SEQ ID NO: 5, A represents the amino acidsequence according to SEQ ID NO: 3 and Q represents the amino acidsequence according to SEQ ID NO:
 4. 6. The nanocapsule of spider silkproteins of claim 1, wherein the nanocapsule comprises pharmaceuticalagents, cosmetical agents, foodstuffs or food additives.
 7. Thenanocapsule of spider silk proteins of claim 6, wherein thepharmaceutical agents, cosmetical agents, foodstuffs or food additivesare encapsulated in the nanocapsule.
 8. The nanocapsule of spider silkproteins of claim 6, wherein the nanocapsule is coated by thepharmaceutical agents, cosmetical agents, foodstuffs or food additives.9. A microcapsule of spider silk proteins, wherein the spider silkproteins form a polymer network that encapsulates an emulsion droplet.10. The microcapsule of spider silk proteins of claim 9, wherein saidmicrocapsule has a wall.
 11. The microcapsule of spider silk proteins ofclaim 10, wherein the wall thickness of said microcapsule is between 5and 100 nm.
 12. The microcapsule of spider silk proteins of claim 9,wherein the spider silk proteins comprise 5 to 50 repeat units, whereinthe repeat unit is selected from the group consisting of the amino acidsequence according to SEQ ID NO: 3, the amino acid sequence according toSEQ ID NO: 4 and the amino acid sequence according to SEQ ID NO:
 5. 13.The microcapsule of spider silk proteins of claim 9, wherein the spidersilk proteins are selected from the group consisting of C₁₆, C₃₂,(AQ)₁₂, (AQ)₂₄, (QAQ)₈ and (QAQ)₁₆, wherein C represents the amino acidsequence according to SEQ ID NO: 5, A represents the amino acid sequenceaccording to SEQ ID NO: 3 and Q represents the amino acid sequenceaccording to SEQ ID NO:
 4. 14. The microcapsule of spider silk proteinsof claim 9, wherein said microcapsule comprises pharmaceutical agents,cosmetical agents, foodstuffs or food additives.
 15. The microcapsule ofspider silk proteins of claim 14, wherein the pharmaceutical agents,cosmetical agents, foodstuffs or food additives are encapsulated in themicrocapsule.
 16. The nanocapsule of spider silk proteins of claim 14,wherein the microcapsule is coated by the pharmaceutical agents,cosmetical agents, foodstuffs or food additives.
 17. A pharmaceuticalcomposition containing the nanocapsule of claim 1 and a pharmaceuticallyacceptable carrier.
 18. A pharmaceutical composition containing themicrocapsule of claim 9 and a pharmaceutically acceptable carrier.
 19. Acosmetical or food product containing the nanocapsule of claim
 1. 20. Acosmetical or food product containing the microcapsule of claim 9.